The cytoskeletal changes that alter cellular morphogenesis and motility depend upon a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. The Rho family of GTP binding proteins is important upstream mediators of cytoskeletal organization. Gem and Rad are members of another family of small GTP binding proteins (the RGK family) for which physiological and biochemical functions have been mostly unknown. Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) a and b. Expression of Gem inhibited ROKb-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, resulting in loss of actinomyosin contractility. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In epistasis assays, Gem opposed ROKb- and Rad opposed ROKa-mediated cell rounding and neurite retraction. In epithelial or fibroblastic cells, Gem or Rad expression resulted in stress fiber and focal adhesion disassembly. In addition, Gem reverted the anchorage-independent growth and invasiveness of Dbl-transformed fibroblasts. These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated via Rho kinase.